Project 3: Overcoming Genetic Variation in Vaccination Abstract. Studies in different human populations produce disparate estimates of protection conferred by BCG against TB. BCG efficacy is >75% in several populations; in contrast, no significant protection is detected in regions where TB is endemic. Many studies find that immunity to mycobacterial infection or BCG is heritable; but, the role of host genetic variation in the success or failure of vaccination is more difficult to quantify. The Collaborative Cross (CC) is a large panel of recombinant inbred mouse lines derived from 8 genetically diverse founder strains, and is reported to capture nearly 90% of the variation present in laboratory mice. Our preliminary study using 8 founder and 3 CC lines found host genetic diversity to be a crucial factor that affects whether BCG vaccination induces protective immunity. As in human meta-analyses of BCG efficacy, when these genetically diverse mice were considered as a single population, BCG has a modest protective effect. However, when assessed by genotype, we found some lines to be protected, others that were not protected, and a few in which vaccination exacerbated disease. These observations indicate that host genetic variation limits BCG efficacy, and suggests that current efforts to develop vaccines that are effective in C57BL/6 mice (i.e., a single genotype), could fail in genetically diverse populations. This project will characterize the genetic and immunological factors that correlate with BCG-induced protection in the CC panel. Aim 1 will extend our preliminary studies to 55 unique CC mouse lines. We will to map the QTLs associated with BCG-elicited protection and in collaboration Project 1, extend the QTL mapping of Mtb susceptibility to the aerosol model. In addition to using bacterial burden as an endpoint, extensive immunological data will be collected. In Aim 2, we will assess 3 different immune states: a) nave; b) BCG vaccinated; or c) vaccinated and Mtb challenged; by classic immune assays, RNA- Seq, and assays based on the recognition of infected cells. With these data, we will identify candidate genes and immune markers that correlate with the QTLs that are associated with BCG-elicited protection. In collaboration with Core B, we will leverage existing samples from the large SATVI cohort of BCG-vaccinated infants to determine if the biomarker profiles or genetic variants that were associated with BCG-efficacy in mice are similarly associated with TB risk, BCG immunogenicity, or efficacy in children. Aim 3 will determine whether CC lines that are not protected by BCG can be used to screen other vaccines. Similarly, we will determine whether CC lines that have a greater dynamic range of protection (more than C57BL/6 mice) could be used to discriminate between different vaccines. Thus, this project seeks to understand the genetic and immunological determinants of vaccine-induced immunity in genetically diverse populations with the ultimate goal of improving the mouse model for pre-clinical development of TB vaccines.